Compound and use thereof in treating autoimmune diseases

ABSTRACT

A compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be used for treating or preventing an autoimmune disease or a cancer. The autoimmune disease may be any one selected from the group consisting of inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, systemic lupus erythematous and type 1 diabetes. The cancer may be selected from the group consisting of colon cancer, melanoma, liver cancer, gliocytoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, kidney cell cancer, stomach cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer and lung cancer.

CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

This application claims the benefit under 35 USC § 119 of Korean PatentApplication Nos. 10-2022-0075726 filed on Jun. 21, 2022 and10-2023-0018717 filed on Feb. 13, 2023 in the Korean IntellectualProperty Office, the entire disclosure of which is incorporated hereinby reference for all purposes.

BACKGROUND 1. Field of the Invention

The present invention relates to a novel compound and use thereof fortreatment of autoimmune diseases.

2. Description of the Related Art

The human body can be protected from pathogens through immune response.Biological defense mechanisms against foreign microorganisms such asviruses and bacteria are normally divided into innate immunity andadaptive immunity, which are mediated by cytokines mostly secreted fromimmune-related cells.

The immune system serves to protect the body from antigens, that is,harmful foreign substances. Types of these antigens include bacteria,viruses, toxins, cancer cells, and blood and tissues from other humansor animals. The immune system produces antibodies and cytokines todestroy these harmful substances. If there are autoimmune disorders, theimmune system cannot distinguish between its body organs and harmfulantigens, and may destroy normal tissues. Diseases derived through sucha response as described above refer to an autoimmune disease.

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcriptionfactor belonging to PER-ARNT-SIM (PAS) superfamily, and is mainlyexpressed in immune cells, epithelial cells, endothelial cells, andstromal cells of barrier tissues. AHR is an environmental sensor anddetects not only xenobiotic ligands such as environmental pollutants(e.g., dioxins), but also physiological ligands generated from cells,microorganisms, and food.

The inactivated form of AHR forms a complex with Hsp90:XAP2:p23:Srcchaperone (AHR chaperone complex) in the cytoplasm, and maintains astructure with high affinity for ligand. When AHR is activated afterligand binding, the complex moves to the nucleus and the AHR isseparated from a chaperone complex and binds to AHR-responsive DNAelements (xenobiotic response elements, XREs) located in the upstreamregulatory regions of a target gene to regulate the expression of thetarget gene. Non-toxic immunomodulatory ligands that can activate AHR invivo may be developed as anew therapeutic agent for autoimmune diseases.

SUMMARY

An aspect of the present invention is to provide a novel compound, astereoisomer or a pharmaceutically acceptable salt thereof.

In addition, another aspect of the present invention is to provide anovel compound useful for prevention and treatment of autoimmunediseases, a stereoisomer or a pharmaceutically acceptable salt thereof.

Further, another aspect of the present invention is to provide apharmaceutical composition for prevention or treatment of autoimmunediseases, including a novel compound, a stereoisomer or apharmaceutically acceptable salt thereof.

To achieve the above aspects, the following technical solutions areadopted in the present invention.

-   -   1. A compound represented by Formula 1 below, a stereoisomer        thereof or a pharmaceutically acceptable salt thereof:

-   -   (wherein, A is hydrogen, halogen, a hydroxyl group, a C₁-C₃        alkyl group, a C₂-C₃ alkenyl group, a C₂-C₃ alkynyl group, a        C₁-C₃ alkoxy group, dimethylamine, —NO₂, —CN, —COOR₂ or        —S(═O)₂R₂,    -   B is hydrogen, a C₁-C₃ alkyl group, a phenyl group, an acetyl        group, —CH₂C(═O)OR₂, —C(═O)OR₂ or —S(═O)₂R₂,    -   R₁ is a substituted or unsubstituted 5-7 membered heterocyclic        ring or —NH₂, and    -   R₂ is a C₁-C₃ alkyl group).    -   2. The compound, the stereoisomer or the pharmaceutically        acceptable salt thereof according to the above 1, wherein the        substituted 5-7 membered heterocyclic ring is a 5-7 membered        heterocyclic ring substituted with a C₁-C₃ alkyl group, a        hydroxyl group or dimethylamine.    -   3. The compound, the stereoisomer or the pharmaceutically        acceptable salt thereof according to the above 1, wherein the        substituted or unsubstituted 5-7 membered heterocyclic ring is        any one selected from the group consisting of the following        heterocyclic rings:

-   -   4. The compound, the stereoisomer or the pharmaceutically        acceptable salt thereof according to the above 1, wherein the        compound represented by Formula 1 is any one selected from the        group consisting of the following compounds.

Compound Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

-   -   5. A pharmaceutical composition, including the compound, the        stereoisomer or the pharmaceutically acceptable salt thereof        according to any one of the above 1 to 4.    -   6. The pharmaceutical composition according to the above 5,        wherein the composition is used for treating or preventing        autoimmune diseases.    -   7. The pharmaceutical composition according to the above 5,        wherein the autoimmune disease is any one selected from the        group consisting of inflammatory bowel disease, multiple        sclerosis, graft-versus-host disease, asthma, atopy, psoriasis,        rheumatoid arthritis, systemic lupus erythematous and type 1        diabetes.    -   8. The pharmaceutical composition according to the above 5,        wherein the composition is used for treating or preventing        cancer.    -   9. The pharmaceutical composition according to the above 8,        wherein the cancer is selected from the group consisting of        colon cancer, melanoma, liver cancer, gliocytoma, ovarian        cancer, colorectal cancer, head and neck cancer, bladder cancer,        kidney cell cancer, stomach cancer, breast cancer, metastatic        cancer, prostate cancer, gallbladder cancer, pancreatic cancer,        blood cancer, skin cancer and lung cancer.    -   10. A method for treatment of autoimmune diseases, including        administering the compound according to any one of the above 1        to 4 to a subject in need thereof.    -   11. A method for treatment of a cancer, including administering        the compound according to any one of the above 1 to 4 to a        subject in need thereof.

The novel compound, the stereoisomer or the pharmaceutically acceptablesalt thereof according to an embodiment of the present invention mayinduce activity of AHR as an immunomodulatory transcription factor,thereby attaining effects of not only controlling inflammation but alsorestoring immune balance and damaged tissues.

The novel compound, the stereoisomer or the pharmaceutically acceptablesalt thereof according to an embodiment of the present invention mayinhibit production of IL-6 as an inflammatory factor, thereby attainingeffects of regulating excessive immune response, in particular,autoimmune response.

The novel compound, the stereoisomer or the pharmaceutically acceptablesalt thereof according to an embodiment of the present invention mayexhibit effects of inducing activity of a regulatory T cell (Treg).

Further, the novel compound, the stereoisomer or the pharmaceuticallyacceptable salt thereof according to an embodiment of the presentinvention may exhibit effects of preventing and treating autoimmunediseases by regulating the above inflammatory factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate measurement of AHR-target gene CYP1A1expression level in order to confirm AHR ligand under cell cultureconditions of a compound of an embodiment of the present invention.

FIG. 3 illustrates measurement of inflammatory factor IL-6 productioninhibitory effects of the compound of an embodiment of the presentinvention.

FIG. 4 illustrates measurement of Foxp3+ regulatory T cell productionpromotion effects of the compound of an embodiment of the presentinvention.

FIGS. 5 and 6 illustrate inflammatory bowel disease therapeutic effectsof the compound of an embodiment of the present invention in an animalmodel with dextran sodium sulfate (DSS)-induced inflammatory boweldisease. Specifically, FIG. 5 demonstrates that the lower the severityindex (Disease Activity Index), the more the treatment is completed,than the control (vehicle), while FIG. 6 shows that the smaller theweight of the colon as compared to the length thereof, the higher thetherapeutic effects of inflammatory bowel disease are.

FIGS. 7 to 9 illustrate effects of the compound of an embodiment of thepresent invention on inhibiting the expression of inflammatory factors(IL-1β, IL-6, IL-17a, TNF-α, S100a8, and S100a9) (FIG. 7 ), increasingthe expression of immunomodulatory factors (IL-10, and Foxp3) (FIG. 8 ),and increasing the expression of intestinal epithelial cell protectivefactors (Reg3b, and Muc2) (FIG. 9 ) in an animal model with DSS-inducedinflammatory bowel disease.

FIG. 10 illustrates mucosal healing effects of the compound of anembodiment of the present invention using FITC-dextran in an animalmodel with DSS-induced inflammatory bowel disease. Herein, it means thatthe lower the detection degree, the higher the mucosa healing effectsare.

FIGS. 11 and 12 illustrate effects of the compound of an embodiment ofthe present invention on preventing colon cancer in AOM/DSS-colorectalcancer animal models, and specifically, show changes in the body weight(FIG. 11 ) and the number and size of tumors (FIG. 12 ) of mice (Normal)not administered with a colorectal cancer-inducing drug (AOM/DSS), mice(DSS+Vehicle) administered with AOM/DSS, and mice (DSS+Compound 6)administered with Compound 6 at the time of AOM/DSS administration.

FIGS. 13 and 14 illustrate effects of the compound of an embodiment ofthe present invention on treating colon cancer in AOM/DSS-colorectalcancer animal models, and specifically, show changes in the body weight(FIG. 13 ) and the number and size of tumors (FIG. 14 ) of mice (Normal)not administered with AOM/DSS, mice (DSS+Vehicle) administered withAOM/DSS, and mice (DSS+Compound 6) administered with Compound 6 aftercolon cancer was induced.

FIGS. 15 to 17 illustrate effects of the compound of an embodiment ofthe present invention on treating psoriasis in animal models, andspecifically, show effects of decreasing epithelial thickness (FIG. 15), effects of inhibiting the expression of skin lesion inflammatorypromoting factors (IL-17a, and S100a8) (FIG. 16 ) and effects ofpromoting the expression of inflammatory suppressing factors (Foxp3, andIL-10) (FIG. 17 ).

FIGS. 18 and 19 illustrate effects of the compound of an embodiment ofthe present invention on treating a graft-versus-host disease (GVHD) inB6→BDF1 animal model, and specifically, show the survival rate of mouseafter transplantation and clinical score (FIG. 18 ), and pathologicalscore (FIG. 19 ).

FIG. 20 illustrates effects of the compound of an embodiment of thepresent invention on treating multiple sclerosis in an autoimmuneencephalomyelitis (EAE) model. Herein, it means that the lower theclinical score, the higher the treating effects are.

FIG. 21 illustrates effects of the compound of an embodiment of thepresent invention on treating neutrophilic asthma in a neutrophilicasthma model. Herein, it means that the lower the number of neutrophilsin bronchoalveolar lavage (BAL), the higher the treating effects are.

FIG. 22 illustrates changes in blood concentration of Compound 6 of anembodiment of the present invention and Comparative Example 1 over timeafter oral administration (PO) or intravenous administration (IV)thereof.

FIG. 23 illustrates effects of Compound 6 of an embodiment of thepresent invention and Comparative Example 1 on inhibiting the activityof Th17 cells producing IL-17A after treatment therewith.

FIG. 24 illustrates effects of Compound 6 of an embodiment of thepresent invention and Comparative Example 1 on promoting the generationof regulatory T cells expressing Foxp3 after treatment therewith.

FIG. 25 illustrates effects of Compound 6 of an embodiment of thepresent invention and Comparative Example 1 on decreasing IL-6 andS100a9 mRNA expression levels after oral administration thereof to micewith inflammatory bowel disease, respectively.

DETAILED DESCRIPTION

Hereinafter, exemplary embodiments of the present invention will bedescribed in detail.

All technical terms used in the present invention are used in the samemeaning as those skilled in the art may generally understand in therelated field of the present invention unless otherwise defined.Further, although preferred methods or samples will be described in thepresent specification, those similar or equivalent are also included inthe scope of the present invention.

In the formula (structural formula) of the present invention, if anysubstituent is not indicated in a site although the site needs asubstituent, it means that a hydrogen substituent is omitted, whichwould be applied to all formulae (structural formulae) in the presentinvention.

An embodiment of the present invention relates to a compound representedby Formula 1 below, a stereoisomer or a pharmaceutically acceptable saltthereof:

In the above Formula 1, A is hydrogen, halogen, a hydroxyl group, aC₁-C₃ alkyl group, a C₂-C₃ alkenyl group, a C₂-C₃ alkynyl group, a C₁-C₃alkoxy group, dimethylamine, —NO₂, —CN, —COOR₂ or —S(═O)₂R₂, B ishydrogen, a C₁-C₃ alkyl group, a phenyl group, an acetyl group,—CH₂C(═O)OR₂, —C(═O)OR₂ or —S(═O)₂R₂, R₁ is a substituted orunsubstituted 5-7 membered heterocyclic ring or —NH₂, and R² is a C₁-C₃alkyl group.

R₁ is substituted on any one of benzene ring carbon atoms ofbenzothiazole.

The 5-7 membered heterocyclic ring may be a compound in which 1 to 2carbon atoms in the ring are substituted with nitrogen or oxygen atoms.

The substituted 5-7 membered heterocyclic ring may be a compound havinga 5-7 membered heterocyclic ring substituted with a C₁-C₃ alkyl group, ahydroxyl group or dimethylamine.

The substituted or unsubstituted 5-7 membered heterocyclic ring may beany one selected from the group consisting of the following heterocyclicrings:

The compound represented by Formula 1 above may be any one selected fromthe group consisting of compounds listed in Table 1 below:

TABLE 1 Compound Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

The compound represented by Formula 1 may be any one selected from thegroup consisting of the following compounds:

-   2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide,    Compound 6);-   2-(5-chloro-1H-indol-3-yl)-N-(6-(4-methylpiperazine-1-yl)benzo[d]thiazol-2-yl)acetamide,    Compound 9);-   2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide,    Compound 21);-   2-(5-hydroxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide,    Compound 32); and-   2-(5-methoxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide,    Compound 39).

Further, an embodiment of the present invention relates to apharmaceutical composition which includes the above-described compound,the stereoisomer or the pharmaceutically acceptable salt thereof.

The pharmaceutical composition may be a pharmaceutical composition fortreatment or prevention of autoimmune diseases. Specifically, thedisease may be inflammatory bowel disease (IBD), multiple sclerosis(MS), graft-versus-host disease (GVHD), asthma, atopy, psoriasis,rheumatoid arthritis (RA), systemic lupus erythematous (SLE), type 1diabetes mellitus (T1D), Behcet's disease or Sjogren's syndrone. Morespecifically, the disease may be inflammatory bowel disease, multiplesclerosis, graft-versus-host disease, asthma atopy, psoriasis,rheumatoid arthritis, systemic lupus erythematous, type 1 diabetes, butit is not limited thereto.

In an embodiment of the present invention, the “autoimmune disease” maycause damage to cells or tissues by humoral immunity, cellular immunityor both thereof. That is, the autoimmune disease is a disease in whichan immune system causes improper reaction to autoantigen thus inducingautoimmune response systemically or specifically in specific organs,etc., which may cause chronic inflammation.

The “inflammatory bowel disease” refers to a disease in which abnormalchronic inflammation in the intestine repeats improvement andrecurrence, and may correspond to one selected from the group consistingof Chron's disease, ulcerative colitis and intestinal Bechet's disease,but it is not limited thereto.

The “multiple sclerosis” refers to an inflammatory disease inducingdemyelination and scar formation as a sign and symptom in a broad sense,which is caused by damage and/or consumption of fatty myelin sheathssurrounding axons of the brain and spinal cord. Types of the multiplesclerosis may include recurrent palliative multiple sclerosis (RRMS),secondary progressive multiple sclerosis (SPMS), primary progressivemultiple sclerosis (PPMS), and progressive recurrent multiple sclerosis(PRMS), but they are not limited thereto.

The “graft-versus-host disease” is a disease in which lymphocytestransfused during hematopoietic stem cell transplantation attack a hostwith deteriorated immune function to cause symptoms such as fever, rash,and abnormalities of liver function, etc., and may invade the skin,lungs, intestines, liver, or the like, but it is not limited thereto.

The “asthma” refers to a disease in which symptoms such as cough andbreathing difficulty occur repeatedly due to inflammation of the bronchiwhen exposed to a specific causative agent, and may be caused byinfection, smoking, allergens, etc., but it is not limited thereto.

The “atopy” refers to atopic dermatitis, and is a representativeallergic disease in which symptoms such as itching and dry skin appearas a chronic recurrent inflammatory skin disease.

The “psoriasis” refers to an inflammatory disease that occurs in theskin or joints due to abnormality in the immune system, and may causeproblems such as an occurrence of ugly appearance, increased keratin, orerythematous plaques, and accompanying pam. The psoriasis may includeany one or more diseases selected from psoriatic arthritis, guttatepsoriasis, pustular psoriasis, red skin psoriasis, scalp psoriasis, nailpsoriasis and enthesitis.

The “rheumatoid arthritis” refers to a systemic autoimmune diseasecharacterized by chronic inflammation of the joint site.

The “systemic lupus erythematous” is also called as “lupus,” and refersto a systemic disease that invades various organs of the body, such asconnective tissue, skin, joints, blood and kidneys, as a chronicinflammatory autoimmune disease. The exact cause is not known, butaccording to previous studies, it is known that genetic factors areassociated with the occurrence of this disease. To help diagnose lupus,the American College of Rheumatology (ACR) has published 11 symptoms,signs, and test findings to help differentiate this disease from otherdiseases. According to the published study, if four or more among the 11symptoms occur, it could be diagnosed as lupus.

The “type 1 diabetes” is an immune-mediated disease in whichinsulin-secreting beta cells are destroyed by an autoimmune reaction.The causes of this disease may include a number of genetic andenvironmental factors, which are specifically targeted toinsulin-secreting beta cells. This disease may be accompanied byprogressive inflammatory infiltration of the pancreatic islets by theimmune cells.

In an embodiment of the present invention, the pharmaceuticalcomposition may be prepared using a pharmaceutically suitable andphysiologically acceptable additive in addition to the activeingredient, which is the compound of an embodiment of the presentinvention. The composition may be administered to a mammal. As theadditive described above, for example, excipients, disintegrants,sweeteners, binders, coating agents, swelling agents, lubricants,glidants or flavoring agents may be used.

Further, the pharmaceutical composition of an embodiment of the presentinvention may be preferably formulated as a pharmaceutical compositionthat includes at least one pharmaceutically acceptable carrier inaddition to the active ingredient in a pharmaceutically effective amountdescribed above for administration.

The “pharmaceutically effective amount” means an amount sufficient totreat a disease with a reasonable benefit/risk ratio applicable tomedical treatment, and an effective dose level may be determined basedon a type and severity of patient's disease, drug activity, drugsensitivity, time of administration, route of administration and rate ofexcretion, duration of treatment, factors including drugs usedconcurrently, and other factors well known in the medical field. Thepharmaceutical composition according to an embodiment of the presentinvention may be administered as an individual therapeutic agent oradministered in combination with other therapeutic agents. Further, thecomposition may be administered sequentially or simultaneously with aconventional therapeutic agent, and may be administered in single ormultiple doses. In consideration of all of the above factors, it isimportant to administer a minimum amount capable of attaining themaximum effect without side effects, such an amount could be easilydetermined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical compositionaccording to an embodiment of the present invention may vary dependingon an age, sex, condition and/or body weight of the patient, absorptionof the active ingredient in the body, inactivation rate and excretionrate, type of disease, and the drug to be used in combination.Typically, 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of bodyweight may be administered daily or every other day, or may be dividedinto 1 to 3 times a day. However, the dosage may be increased ordecreased depending on the route of administration, severity of obesity,sex, body weight, age, etc., therefore, would not limit the scope of thepresent invention in any way.

Further, the “pharmaceutically acceptable” refers to a composition thatis physiologically acceptable and does not usually cause allergicreactions such as gastrointestinal disorders and dizziness, or similarreactions when administered to humans.

Examples of the carrier, excipient and diluent may include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, gum acacia, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water,methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesiumstearate, and mineral oils. Further, fillers, anti-aggregating agents,lubricants, wetting agents, flavoring agents, emulsifying agents, andpreservatives may additionally be included.

Further, the composition of an embodiment of the present invention maybe formulated using any method known in the art in order to providerapid, sustained or delayed release of the active ingredient afteradministration thereof to a subject in need of treatment using thepharmaceutical composition of an embodiment of the present inventionincluding humans. The formulation may be powder, granule, tablet,emulsion, syrup, aerosol, soft or hard gelatin capsule, sterileinjectable solution, or sterile powder.

The expression “pharmaceutically acceptable salt” refers to a saltprepared using a specific compound according to the present invention,as well as acid or base relatively nontoxic thereto. Thepharmaceutically acceptable salt may include, for example, acid additionsalts or metal salts.

The acid addition salts may be formed from inorganic acids such ashydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid,aliphatic mono and dicarboxylates, phenyl-substituted alkanoates,hydroxy alkanoates and alkane dioates, and non-toxic organic acids suchas aromatic acids, aliphatic and aromatic sulfonic acids. Thesepharmaceutically non-toxic salts may include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride,bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate,acrylate, formate, isobutyrate, caprate, heptanoate, propyolate,oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate,butine-1,4-dioate, nucleic acid-1,6-dioate, benzoate, chlorobenzoate,methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,phthalate, terephthalate, benzene sulfonate, toluene sulfonate,chlorobenzene sulfonate, xylene sulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, β_hydroxybutyrate,glycolate, malate, tartrate, methane sulfonate, propane sulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The metal salt may be a sodium, potassium or calcium salt. The metalsalt may be prepared using a base, for example, alkali-metal or alkalineearth metal salts may be obtained by dissolving the compound in anexcess amount of alkali-metal hydroxide or alkaline earth metalhydroxide solution, filtering the non-dissolved compound salt, andevaporating or drying the filtrate.

An embodiment of the present invention nay relate to a method fortreatment of an autoimmune disease, which includes administering thecompound, the stereoisomer or the pharmaceutically acceptable saltthereof to a subject in need thereof.

Further, an embodiment of the present invention may relate to a methodfor inducing activity of AHR, which includes administering the compound,the stereoisomer or the pharmaceutically acceptable salt thereof.

Specifically, the compounds of an embodiment of the present inventionmay target the aryl hydrocarbon receptor (AHR), which is animmunomodulatory transcription factor of an embodiment of the presentinvention, and may serve as an agent to induce AHR activity, therebycontrolling inflammation, regulating immune balance, and repairingdamaged tissue. Therefore, the compound may be used for therapeuticpurposes of autoimmune diseases, but it is not limited thereto. Existingligands are toxic, have low affinity and structural stability, and hightarget non-specificity, which entail a problem in that these areunsuitable for development into pharmaceutical compositions. On theother hand, when AHR activity is induced by the compound of anembodiment of the present invention having “drug-like properties,” itcould be effectively used for treatment and prevention of autoimmunediseases.

An embodiment of the present invention may relate to a method forinhibiting production of IL-6, which includes administering thecompound, the stereoisomer or the pharmaceutically acceptable saltthereof.

Specifically, since the inflammatory factor IL-6 is known to causeautoimmune diseases, the compound of the present invention may be usedin treatment of autoimmune diseases through a mechanism that inhibitsthe production thereof. Actually, there is a number of known therapeuticagents for autoimmune diseases that target inhibition of IL-6, as wellas related papers. According to the following experimental data, thecompound of an embodiment of the present-invention is also confirmed toinhibit the production of IL-6 and thus is expected to have effects ofreducing the autoimmune response, whereby the composition of anembodiment of the present invention may be used for treatment andprevention of autoimmune diseases.

Further, an embodiment of the present invention relates to a compositionfor prevention or treatment of a cancer, which includes the compound,the stereoisomer or the pharmaceutically acceptable salt thereof.

In the present invention, “cancer” broadly refers to uncontrolledabnormal growth of the host's own cells that invade the surroundingtissues of the initial abnormal cell growth site in the host andpotential tissues located distally of these sites. Further, carcinoma asa cancer of epithelial tissues (e.g. the skin, squamous cells); sarcoma,as a cancer of connective tissue (e.g. bone, cartilage, fat, muscle,blood vessels, etc.); leukemia as a cancer of blood-forming tissue(e.g., bone marrow tissue); lymphoma and myeloma, which are cancers ofimmune cells; cancers of the central nervous system, including cancersin the brain and spinal tissue, may be included.

Specifically, the cancer may be selected from the group consisting ofcolon cancer, melanoma liver cancer, gliocytoma, ovarian cancer,colorectal cancer, head and neck cancer, bladder cancer, kidney cellcancer, stomach cancer, breast cancer, metastatic cancer, prostatecancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancerand lung cancer, but it is not limited thereto.

An embodiment of the present invention relates to a method for treatmentof a cancer, which includes administering the compound, the stereoisomeror the pharmaceutically acceptable salt thereof to a subject in needthereof.

The treatment method may include administering the compound, thestereoisomer or the pharmaceutically acceptable salt thereof to apatient who was diagnosed with cancer, at any stage of chemotherapy, andit is not limited to a specific stage.

Further, the compound, the stereoisomer or the pharmaceuticallyacceptable salt thereof may be administered in the aforementioned formsof the pharmaceutical composition, but it is not limited thereto.

The compound represented by Formula 1 according to an embodiment of thepresent invention may be prepared by any method known in variousdocuments.

Hereinafter, the present invention will be described in detail by meansof preparative examples and examples of the present invention.

PREPARATIVE EXAMPLE Preparative Example 1: Synthesis of2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinebenzo[d]thiazol-2-yl)acetamide(Compound 6)

While stirring a solution of 2-(5-chloro-1H-indol-3-yl)acetic acid(56.00 g, 267.14 mmol), 2,3,4,5,6-pentafluorophenol (54.09 g, 293.85mmol), and EDC-HCl (61.45 g, 320.57 mmol) in dichloromethane (890 mL) atroom temperature, N,N-diisopropylethylamine (52.82 mL, 293.85 mmol) wasadded. After stirring the reaction mixture at room temperature for 24hours and confirming the completion of the reaction, distilled water(890 mL) was added. The layers were separated, and the organic layer wasdried over anhydrous Na₂SO₄ and filtered. After concentrating thefiltrate under reduced pressure, the concentrate was purified by columnchromatography to yield perfluorophenyl2-(5-chloro-1H-indol-3-yl)acetate (50.00 g, yield 50%).

A solution of 6-morpholinobenzo[d]thiazol-2-amine (26.00 g, 110.49mmol), perfluorophenyl 2-(5-chloro-1H-indol-3-yl)acetate (47.74 g,127.07 mmol) in THF (1.1 L) was stirred. The reaction mixture wasrefluxed for 24 hours and completion of the reaction was confirmed,followed by cooling it to room temperature. The reaction mixture wasconcentrated under reduced pressure and purified by MPLC to yieldCompound 6 (28.5 g, yield 60%).

¹H NMR (DMSO-d6, 400 MHz): δ 12.37 (s, 1H), 11.12 (s, 1H), 7.59 (t, 2H,J=16.0 Hz), 7.43 (d, 2H, J=16.0 Hz), 7.34 (s, 1H), 7.12 (d, 1H, J=8.0Hz), 7.02 (d, 1H, J=12.0 Hz), 3.88 (m, 2H), 3.74 (m, 4H), 3.11 (m, 4H)

Preparative Example 2: Synthesis of2-(5-chloro-1H-indol-3-yl)-N-(6-(4-methylpiperazin-1-yl)benzo[d]thiazol-2-yl)acetamide(Compound 9)

A solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (210 mg, 1.00 mmol),6-(4-methylpiperazin-1-yl)benzo[d]thiazol-2-amine (249 mg, 1.00 mmol),HBTU (760 mg, 2.00 mmol), and DIPEA (0.7 mL, 4.01 mmol) was added in DMF(10 ml) and stirred for 2 days. After completion of the reaction, thereaction mixture was put into ice-water (50 mL), stirred for 30 minutes,filtered, and washed with water. The obtained solid mixture was purifiedby column chromatography to yield Compound 9 (100 g, 22%).

¹H NMR (DMSO-d6, 400 MHz): δ 12.37 (s, 1H), 11.20 (s, 1H), 7.67 (d, 1H,J=4.0 Hz), 7.56 (d, 1H, J=8.0 Hz), 7.44 (d, 1H, J=4.0 Hz), 7.37 (m, 2H),7.06 (m, 2H), 3.87 (s, 2H), 3.14 (m, 4H), 2.50 (m, 4H), 2.24 (s, 3H)

Preparative Example 3: Synthesis of2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinebenzo[d]thiazol-2-yl)acetamide(Compound 21)

While stirring a solution of 2-(5-chloro-1-methyl-1H-indol-3-yl)aceticacid (110 mg, 491.83 μmol), 2,3,4,5,6-pentafluorophenol (100 mg, 541.01μmol), EDC-HCl (113 mg, 590.19 μmol) in dichloromethane (1.6 mL),N,N-diisopropylethylamine (0.1 mL, 541.01 μmol) was added. Afterstirring the reaction mixture at room temperature for 24 hours andconfirming the completion of the reaction, distilled water (2 mL) wasadded. The layers were separated, and the organic layer was dried overanhydrous Na₂SO₄ and filtered. After concentrating the filtrate underreduced pressure, the concentrate was purified by column chromatographyto yield perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate (150mg, 78%).

A solution of 6-morpholinobenzo[d]thiazol-2-amine (40 mg, 169.99 μmol)and perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate (99 mg,254.99 μmol) in THF (1.7 mL) was stirred at reflux for 12 hours. Thereaction mixture was cooled to room temperature, followed by filteringunder reduced pressure to remove impurities, and the filtrate wasconcentrated under reduced pressure. The concentrated reaction mixturewas recrystallized with diethyl ether to yield Compound 21 (40 mg, yield54%).

¹H NMR (DMSO-d6, 500 MHz): δ 12.38 (s, 1H), 7.69 (s, 1H), 7.58 (d, 1H,J=10.0 Hz), 7.44 (d, 2H, J=5.0 Hz), 7.36 (s, 1H), 7.11 (m, 2H), 3.86 (s,2H), 3.77 (s, 3H), 3.73 (m, 4H), 3.11 (m, 4H)

Preparative Example 4: Synthesis of2-(5-hydroxy-1H-indol-3-yl)-N-(6-morpholinebenzo[d]thiazol-2-yl)acetamide(Compound 32)

Compound 39(2-(5-methoxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide)(25 mg, 59.17 μmol) obtained in Preparative Example 5 below inborontribromide (7.9 μml, 71.01 μmol) was stirred at room temperaturefor 6 hours. Distilled water was added to the reaction mixture toterminate the reaction. After neutralization with NaHCO₃, the layerswere separated with DCM. The organic layer was washed, dried overanhydrous Na₂SO₄ and filtered. After concentrating the filtrate underreduced pressure, the concentrate was recrystallized with diethyl etherto yield Compound 32 (11 mg, yield 45%).

¹H NMR (DMSO-d6, 500 MHz): δ 12.30 (s, 1H), 10.65 (s, 1H), 8.64 (s, 1H),7.57 (d, 1H, J=10.0 Hz), 7.45 (s, 1H), 7.19 (s, 1H), 7.11 (m, 2H), 6.90(s, 1H), 6.59 (d, 1H, J=5.0 Hz), 3.78 (s, 2H), 3.74 (m, 4H), 3.11 (m,4H)

Preparative Example 5: Synthesis of2-(5-methyl-1H-indol-3-yl)-N-(6-morpholinebenzo[d]thiazol-2-yl)acetamide(Compound 39) (1) Synthesis of perfluorophenyl2-(5-methoxy-1H-indol-3-yl)acetate

While stirring a solution of 2-(5-methoxy-1H-indol-3-yl)acetic acid (200mg, 974.61 μmol), 2,3,4,5,6-pentafluorophenol (198 mg, 1.07 mmol), andEDC-HCl (225 mg, 1.17 mmol) in dichloromethane (3.3 mL),(N,N-diisopropylethylamine (0.2 mL, 1.07 mmol), was added. Afterstirring the reaction mixture at room temperature for 24 hours andconfirming the completion of the reaction, distilled water (4 mL) wasadded. The layers were separated, and the organic layer was dried overanhydrous Na₂SO₄ and filtered. After concentrating the filtrate underreduced pressure, the concentrate was purified by column chromatographyto yield the title compound (288 mg, 80%).

A solution of 6-morpholinobenzo[d]thiazol-2-amine (100 mg, 424.98 μmol)and perfluorophenyl 2-(5-methoxy-1H-indol-3-yl)acetate (189 mg, 509.98μmol) in THF (4.3 mL) was stirred at reflux for 12 hours. The reactionmixture was cooled to room temperature, followed by filtering underreduced pressure to remove impurities, and the filtrate was concentratedunder reduced pressure. The concentrated reaction mixture wasrecrystallized with diethyl ether to yield Compound 39 (120 mg, yield67%).

¹H NMR (DMSO-d6, 500 MHz): δ 12.34 (s, 1H), 10.82 (s, 1H), 7.57 (d, 1H,J=10.0 Hz), 7.45 (s, 1H), 7.24 (d, 2H, J=10.0 Hz), 7.11 (m, 2H), 6.72(dd, 1H, J=10.0 Hz), 3.84 (s, 2H), 3.74 (m, 7H), 3.10 (m, 4H)

Comparative Example 1: Synthesis ofN-(benzo[d]thiazol-2-yl)-2-(5-chloro-1H-indol-3-yl)acetamide(Comparative Example 1)

While stirring a solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (7.0g, 33.39 mmol) in DMF (100 mL) at room temperature,benzo[d]thiazol-2-amine (4.51 mg, 30.05 mmol),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxidehexafluorophosphate (HATU, 15.24 g, 40.07 mmol) and trimethylamine (9.4mL, 66.78 mmol) were sequentially added. The reaction mixture wasstirred at room temperature for 3 days. Distilled water was added to themixture to terminate the reaction. The layers were separated with ethylacetate, and the organic layer was washed with distilled water, driedover anhydrous Na₂SO₄, and filtered. After concentrating the filtrateunder reduced pressure, the concentrate was purified by columnchromatography to obtain a compound of Comparative Example 1 (5.0 g,yield 45%).

¹H NMR (DMSO-d6, 400 MHz): δ 12.57 (s, 1H), 11.21 (s, 1H), 7.94 (d,J=8.0 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.68 (d, J=4.0 Hz, 1H), 7.37 (m,3H), 7.27 (t, J=16.0 Hz, 1H), 7.07 (dd, J=8.0 4.0 Hz, 1H), 3.91 (s, 2H)

EXAMPLE Example 1: Confirmation of Effects of Inducing CYP1A1 Expression

In order to confirm target specificity of the compounds of an embodimentof the present invention prepared as above, it was confirmed whether theexpression levels of CYP1A¹ mRNA and CYP1A1 protein, which are AHRtarget genes, were increased.

(1) Effects of Inducing CYP1A1 mRNA Expression

After recovering HepG2 under culture in DMEM-fetal bovine serum (FBS)10% medium and then confirming that the survival rate is 97% or morethrough trypan blue staining, the recovered product was centrifuged at aspeed of 1200 rpm for 5 minutes at room temperature, and the cells wereprepared by resuspending them in DMEM-fetal calf serum 10% medium at3×10⁵ cells/ml. Thereafter, the cells were dispensed onto a 60 mm dishby 3 ml, and each dish was treated with 50 μl of Compounds 6, 21, 32 and39 at a concentration of 1 μM diluted in DMEM medium, and then culturedin a cell incubator (5% CO₂incubator) for 24 hours. As a control, 50 μlof 0.05% dimethylsulfoxide (DMSO)/DMEM medium was used for treatment.

The cultured cells were recovered to prepare an mRNA sample.Specifically, mRNA was extracted from the recovered cells by aphenol-chloroform precipitation method using TRIZOL reagent (Invitrogen,Cat No. 15596018). From the separated RNA, cDNA was synthesized byreverse transcription, and the expression of CYP1A1 was confirmedthrough a real-time polymerase chain reaction (PCR) using iQ SYBR-GreenSupermix (Bio-rad) in a CFX96 (Bio-rad) detection system. Relativevalues of the enzyme expression levels were compared by ΔΔct methodusing GAPDH as a control enzyme. Herein, one (1) fold was set using thecontrol.

The real-time polymerase chain reaction was performed under theconditions of 45 cycles at an annealing temperature of 58° C., whereinthe following primer sequences were used. Human CYP1A¹ forward, 5′-CACCCT CAT CAG TAA TGG TCA GA-3′ (SEQ ID NO: 1) and reverse, 5′-AAC GTG CTTATC AGG ACC TC-3′ (SEQ ID NO: 2); Human GAPDH forward, 5′-TGA TGA CATCAA GAA GGT GG-3′ (SEQ ID NO: 3) and reverse, 5′-TTA CTC CTT GGA GGC CATGT-3′ (SEQ ID NO: 4).

From the above results, it can be confirmed that groups treated withCompounds 6, 21, 32 and 39 showed higher CYP1A1 mRNA expression levelsthan the control (vehicle). Accordingly, it was confirmed that Compounds6, 21, 32 and 39 significantly induced the expression of CYP1A¹, an AHRtarget gene (FIG. 1 ).

(2) Effect of Inducing CYP1A1 Protein Expression

After recovering HepG2 under culture in DMEM-fetal bovine serum (FBS)10% medium and then confirming that the survival rate is 97% or morethrough trypan blue staining, the recovered product was centrifuged at aspeed of 1200 rpm for 5 minutes at room temperature, and the cells wereprepared by resuspending them in DMEM-fetal calf serum 10% medium at1.5×10⁵ cells/ml.

Thereafter, the cells were dispensed onto a 96-well plate by 200 μl, andeach plate was treated with 3 μl of Compounds 6, 21, 32 and 39 at aconcentration of 1 μM diluted in DMEM medium, and then cultured in acell incubator (5% CO₂incubator) for 24 hours. As a control, 3 μl of0.05% dimethylsulfoxide (DMSO)/DMEM medium was used for treatment.

After 24 hours, the medium of the plate was discarded, washed with dPBS,treated with 100 μl of 2 μM 7-ethoxyresorufin, and cultured in a cellincubator (5% CO₂ incubator) for 30 minutes. After 30 minutes, each 75μl of fluorescamine (150 μg/ml) was treated, then resorufine excitationwas measured at 535 nm and emission at 590 nm. The standard curve wasmeasured and used with 0 to 50 pmol of resorufin solution.

After measurement, all solutions were removed, followed by treating thesame with 25 μl of 0.5 M sodium hydroxide (NaOH), the cells were scrapedand reacted in a shaker at room temperature for 15 minutes.

After 15 minutes, protein was quantified using a Bradford method, and avalue was obtained using a calculation formula (sample resorufinamount/sample protein amount), and then one (1) fold was set using thecontrol.

From the above results, it can be confirmed that the CYP1A¹ proteinexpression level is significantly higher in the groups treated withCompounds 6, 21, 32 and 39 than the control (vehicle), and therefore, itwas confirmed that protein of AHR-target gene CYP1A1 was significantlyinduced by Compounds 6, 21, 32 and 39 (FIG. 2 ).

Example 2: Inhibitory Effects of Production of Inflammatory Factor IL-6

In order to assess the inhibitory effects of production of IL-6 by thecompounds of the present invention prepared as above, an IL-6 productiontest of epithelial cells by stimulation of IL-1β was performed.

After recovering A549 cells under culture in DMEM-fetal bovine serum(FBS) 10% medium and confirming that the survival rate is 97% or morethrough trypan blue staining, the recovered product was centrifuged at aspeed of 1200 rpm for 5 minutes at room temperature, and the cells wereprepared by resuspending them in DMEM-10% fetal bovine serum medium at1×10⁶ cells/3 ml. Thereafter, the cells were dispensed by 3 ml onto a 60mm plate, and each dish was treated with 50 μl of Compounds 6, 9, 21, 32and 39 at a concentration of 5 μM diluted in DMEM medium, and thencultured in a cell incubator (5% CO₂ incubator) for 48 hours. Then, thecells were treated with 50 μl of human recombinant IL-1β at aconcentration of 12.5 ng/ml diluted in DMEM medium, followed byculturing the same in a cell incubator (5% CO2incubator) for 24 hours.As a control, 50 μl of 0.05% dimethylsulfoxide (DMSO)/DMEM medium wasused for treatment.

The cultured cells were recovered to prepare an mRNA sample.Specifically, mRNA was extracted from the recovered cells by aphenol-chloroform precipitation method using TRIZOL reagent (Invitrogen,Cat No. 15596018). From the separated RNA, cDNA was synthesized byreverse transcription, and the expression of CYP1A1 was confirmedthrough a real-time polymerase chain reaction (PCR) using iQ SYBR-GreenSupermix (Bio-rad) in a CFX96 (Bio-rad) detection system. Relativevalues of the enzyme expression levels were compared by ΔΔct methodusing GAPDH as a control enzyme. Herein, one (1) fold was set using thecontrol.

The real-time polymerase chain reaction was performed under theconditions of 45 cycles at an annealing temperature of 58° C., whereinthe following primer sequences were used. Human IL-6 forward, 5′-GAT GGCTGA AAA AGA TGG ATGC-3′ (SEQ ID NO: 5) and reverse, 5′-TGG TTG GGT CAGGGG TGG TT-3′ (SEQ ID NO: 6); Human GAPDH forward, 5′-TGA TGA CAT CAAGAA GGT GG-3′ (SEQ ID NO: 3) and reverse, 5′-TTA CTC CTT GGA GGC CATGT-3′ (SEQ ID NO: 4).

As a result, it was confirmed that IL-6 production of A549 by IL-10stimulation was significantly reduced by treatment with Compounds 6, 9,21, 32 and 39, and therefore, IL-6 production was effectively inhibitedby Compounds 6, 9, 21, 32 and 39 (FIG. 3 ).

Example 3: Confirmation of Effects of Promoting Production ofFoxp3-Expressing Regulatory T Cells (Treg)

Effects of promoting the production of “FoxP3-expressing regulatory Tcells,” which play an important role in maintaining immune tolerance,was evaluated (FIG. 4 ).

The spleen of C57BL/6 mice (8 to 12 weeks old, female) was excised,pulverized by adding RPMI medium, and then passed through a 40 Vim cellstrainer (BD Falcon), thereby obtaining a single cell suspension. Thesingle cell suspension was centrifuged (1200 rpm, 5 minutes), and afterdiscarding the supernatant, 1 ml of ACK lysis buffer was added, followedby stirring for 1 minute and then washing the same with RPMI medium.After the single cell suspension was centrifuged, T-cells were separatedusing a Mouse CD4 Naive T cell Enrichment Kit (Invitrogen). Theseparated T-cells were prepared by resuspending them in RPMI-fetalbovine serum (FBS) 10%+2-ME (mercaptoethanol) medium at 1×10⁶/ml. ForT-cell activation, 5 μg/ml anti-CD3 (eBioscience™) was dispensed by 100μl onto a 96-well plate, reacted in a cell incubator (37° C., 5% CO₂incubator) for 4 hours, and washed with phosphate buffered saline toprepare the plate. The T-cells were dispensed by 200 μl onto theprepared plate, and each well was treated with 2 μg/ml of anti-CD28(eBioscience™), 2 ng/ml of TGFβ-1 (R&D systems), and 100 U/ml of IL-2.Each 5 μl of Compounds 6 and 9 at a concentration of 2.5 μM diluted inRPMI-fetal bovine serum 10%+2-ME medium was used for treatment, followedby culturing the same in a cell incubator (37° C., 5% CO₂ incubator) for3 days. As a control, 5 μl of 0.05% dimethylsulfoxide (DMSO)/RPMI mediumwas used for treatment. After 3 days, in order to confirm effects ofproducing regulatory T cells, the cultured cells were recovered tomeasure the expression of Foxp3 protein.

In order to confirm the expression of Foxp3 protein, the recovered cellswere placed in a 5 ml FACS tube (BD Falcon) and washed with 1 ml ofphosphate buffered saline. The cells were resuspended in 0.1 ml of FACSbuffer (0.1% NaN3, 1% FBS) and treated with 0.5 μg of CD16/CD32 antibody(eBioscience™) to prevent non-specific binding of the antibody, thenreacted at 4° C. for 15 minutes. Thereafter, the cells were treated with0.25 μg of CD4 Monoclonal Antibody (GK1.5) and PE-Cyanine7(eBioscience™), and stained at 4° C. for 30 minutes, followed by washingwith 1 ml of FACS buffer. Then, 1 ml of Fixation/Permeabilizationsolution (eBioscience™) was added to the FACS tube containing eachsample, followed by reaction at 4° C. for 1 hour. Then, the product waswashed twice with Permeabilization buffer (eBioscience™). Thereafter,0.5 μg of Foxp3 Monoclonal Antibody (eBioscience™) was used fortreatment, followed by staining the same at 4° C. for 30 minutes. Thecells were washed twice with the Permeabilization buffer, suspended in0.3 ml of FACS buffer, and measured by flow cytometry.

As a result, it was confirmed that the production of Foxp3-expressingregulatory T cells was markedly increased by Compounds 6 and 9 comparedto Vehicle, and therefore, the production of FoxP3+ regulatory T cellswas effectively promoted by Compounds 6 and 9 (FIG. 4 ).

Example 4: Confirmation of Therapeutic Effects of Inflammatory BowelDiseases

In order to investigate the therapeutic effects of the compoundsaccording to the present invention prepared as above on inflammatorybowel disease, the inflammatory bowel disease was induced in C57BL/6mice, and Compound 6 was administered to the mice to evaluate theefficacy as follows (FIGS. 5 to 10 ).

(1) Confirmation of Therapeutic Effects of Compound 6 on InflammatoryBowel Diseases

On day 0 of the experiment, a 2.0% DSS solution prepared by dissolvingDSS (Dextran sulfate sodium, MP biomedicals, Cat No. 160110) in 2.0%sterile distilled water was given for drinking to C57BL/6 mice (11 weeksold, female, 20±2 g) for 7 days. The 2.0% DSS solution was changed at aninterval of 2 days. Sterile distilled water was provided for drinkingfrom the 8th day of the experiment. Body weight and severity index weremeasured at an interval of 2 days from the 0th day of the experiment inorder to confirm the onset of inflammatory bowel disease.

10 mg/kg of Compound 6 per mouse was completely dissolved in anethanol-Cremophor EL mixture corresponding to 7.5% (v/v) of theadministered dose, and then, was diluted in phosphate buffered saline toprepare the final ethanol:Cremophor EL:phosphate buffered saline(0.375:0.375:9.25, v/v/v), followed by oral administration of 200 μldaily for a total of 14 times from the 1st to 14th days of theexperiment. An inflammatory bowel disease severity index was visuallyobserved and recorded at an interval of 2 days according to a severityindex system classified into 0 to 10 levels.

Inflammatory bowel disease symptoms were evaluated by summing the scoresof three items according to the following items (Table 2).

TABLE 2 Symptoms Reduction of Score Watery of excrement Melena stateBody weight 0 Normal form Normal form Normal 1 Slightly loose fecesBrown excrement  5-10% decrease 2 Loose feces Redish brown 11-15%decrease excrement 3 Diarrhea Melena 16-20% decrease 4 — —   >20%decrease

As a result of the analysis, it was confirmed that the body weight ofthe solvent control (Vehicle) started to decrease from the 6th day ofthe experiment, decreased by 10% or more on the 10th day of theexperiment and 100% of enteritis was induced along with an increasedseverity index of 5 or more. The mice in the solvent control showed aseverity index of 7.50±0.50 on the 10th day of the experiment when theseverity index reached the maximum. On the other hand, the experimentalgroup administered with 10 mg/kg of Compound 6 of an embodiment of thepresent invention showed statistically significant therapeutic effectscompared to the solvent control on the 10th day of the experiment.Further, comparison of the colon weight:length ratio on the 15th day ofthe experiment (weight:length ratio, mg/cm) demonstrated that intestinalinflammation could be significantly suppressed in terms of morphology(compared to the solvent control (Vehicle): ***, p<0.001, see FIGS. 5and 6 ). Specifically, Compound 6 of an embodiment of the presentinvention showed excellent anti-inflammatory effects when administeredin an amount of 10 mg/kg.

On the 15th day of the experiment, the colon of the mouse was excised toprepare an mRNA sample. In order to extract mRNA, the colon tissue wasground with a homogenizer to acquire a homogeneous suspension, From thehomogeneous suspension, mRNA was extracted by a phenol-chloroformsedimentation method using an easy-Spin™ (DNA free) total RNA extractionkit (Intron biotechnology. Cat No. 17221). From the isolated RNA cDNAwas synthesized by reverse transcription, followed by confirming theexpression of inflammatory cytokines through real-tire polymerase chainreaction (PCR) using iQ SYBR-Green Supermix (Bio-rad) in the CFX96(Bio-rad) detection system. Relative values of the enzyme expressionlevels were compared by the ΔΔct method using GAPDH as a control enzyme.Herein, one (1) fold was set using the normal mouse colon as a control.

The real-time polymerase chain reaction was implemented under theconditions of 45 cycles at an annealing temperature of 58° C. and thefollowing primer sequences were used.

Mouse IL-10 forward, 5′-CTC GTG CTG TCG GAC CCA TAT-3′ (SEQ ID NO: 7)and reverse, 5′-TTG AAG ACA AAC CGC TTT TCC A-3′ (SEQ ID NO: 8);

Mouse IL-6 forward, 5′-CAT GTT CTC TGC GAA ATC GTG G-3′ (SEQ ID NO: 9)and reverse, 5′-AAC GCA CTA GGT TTG CCG AGT A-3′ (SEQ ID NO: 10);

Mouse IL-17A forward, 5′-TIT AAC TCC CTT GGC GCA AAA-3′ (SEQ ID NO: 11)and reverse, 5′-CTT TCC CTC CGC ATT GAC AC-3′ (SEQ ID NO: 12);

Mouse TNF-α forward, 5′-CCA CAC CGT CAG CCG ATT TG-3′ (SEQ ID NO: 13)and reverse, 5′-CAC CCA TTC CCT TCA CAG AGC-3′ (SEQ ID NO: 14);

Mouse S100a8 forward, 5′-AAA TCA CCA TGC CCT CTA CAA G-3′ (SEQ ID NO:15) and reverse, 5′-CCC ACT TIT ATC ACC ATC GCA A-3′ (SEQ ID NO: 16);

Mouse S100a9 forward, 5′-ATA CTC TAG GAA GGA AGG ACA CC-3′ (SEQ ID NO:17) and reverse, 5′-TCC ATG ATG TCA TTT ATG AGG GC-3′ (SEQ ID NO: 18);

Mouse IL-10 forward, 5′-CAA GGC AGT GGA GCA GGT GAA-3′ (SEQ ID NO: 19)and reverse, 5′-CGG AGA GAG GTA CAA ACG AGG TT-3′ (SEQ ID NO: 20);

Mouse Foxp3 forward, 5′-CCC ATC CCC AGG AGT CTT G-3′ (SEQ ID NO: 21) andreverse, 5′-ACC ATG ACT AGG GGC ACT GTA-3′ (SEQ ID NO: 22);

Mouse Reg3b forward, 5′-ACT CCC TGA AGA ATA TAC CCT CC-3′ (SEQ ID NO:23) and reverse, 5′-CGC TAT TGA GCA CAG ATA CGA G-3′ (SEQ ID NO: 24);

Mouse Muc2 forward, 5′-ATG CCC ACC TCC TCA AAG AC-3′ (SEQ ID NO: 25) andreverse, 5′-GTA GTT TCC GTT GGA ACA GTG AA-3′ (SEQ ID NO: 26);

Mouse GAPDH forward, 5′-TTC ACC ACC ATG GAG AAG GC-3′ (SEQ ID NO: 27)and reverse, 5′-GGC ATG GAC TGT GGT CAT GA-3′ (SEQ ID NO: 28).

The expression levels of the inflammatory cytokines IL-1β, IL-6, IL-17A,TNF-α, S100a8, and S100a9 in colon lesions were significantly reducedcompared to the solvent control (Vehicle) by administration of Compound6 (compared to the solvent control (Vehicle): **, p<0.01, see FIG. 7 ).In addition, the expression levels of the immunomodulatory factors IL-10and Foxp3 in colon lesions were significantly increased compared to thesolvent control (Vehicle) by administration of Compound 6 (compared tothe solvent control (Vehicle): *, p<0.05; **, p<0.01, see FIG. 8 ).Further the expression levels of intestinal epithelial cell protectivefactors Reg3b and Muc2 in colon lesions were significantly increasedcompared to the vehicle control (Vehicle) by administration of Compound6 (compared to the solvent control (Vehicle): *, p<0.05; ***, p<0.001,see FIG. 9 ). From these results, it could be seen that Compound 6 of anembodiment of the present invention can significantly reduce theexpression of inflammatory factors in the intestine, while significantlyincreasing the expressions of intestinal immunomodulatory factors andintestinal epithelial cell protective factors.

In order to investigate mucosal healing effects of the compoundsaccording to an embodiment of the present invention, inflammatory boweldisease was induced in C57BL/6 mice, and a degree of recovery ofintestinal epithelial barrier integrity was assessed by administeringCompound 6 as follows.

On day 0 of the experiment, a 20% DSS solution prepared by dissolving2.0% DSS in sterile distilled water was given for drinking to C57BL/6mice (11 weeks old, female, 20±2 g) for 7 days. The 2.0% DSS solutionwas changed at an interval of 2 days. Sterile distilled water wasprovided for drinking from the 8th day of the experiment. Body weightand severity index were measured at an interval of 2 days from the 0thday of the experiment, so as to confirm the onset of inflammatory boweldisease.

In the compound 6 administration group according to an embodiment of thepresent invention, 10 mg/kg of the compound per mouse was completelydissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v/v)of the administered dose, and then, was diluted in phosphate bufferedsaline to prepare the final ethanol:Cremophor EL (0.375:0.375:9.25,v/v/v), followed by oral administration with 200 μl of the preparedsolution daily for a total of 14 times from the 1st to 14th days of theexperiment.

One day before FITC-dextran administration, a mouse was deprived ofwater overnight. On the 15th day of the experiment, 600 ng/kg ofFITC-dextran (Fluorescein isothiocyanate-dextran, Sigma Aldrich, Cat No.FD40) was diluted in a phosphate buffered saline and administered orallyto the mouse at 200 μl once, 4 hours after oral administration,fluorescence was measured in the serum extracted from the heart(fluorometer, excitation 485-490 nm, emission 528-530 nm).

Serum FITC-dextran was significantly decreased compared to the solventcontrol (Vehicle) by administration of Compound 6 (compared to solventcontrol (Vehicle): ***, p<0.001, see FIG. 10 ). From the above resultsresult, it could be seen that Compound 6 of an embodiment of the presentinvention showed significant mucosal healing effects. From the aboveresults, it was confirmed that Compound 6 of an embodiment of thepresent invention has oral administration therapeutic effects in a mousemodel of inflammatory bowel disease.

Example 5: Confirmation of Effects of Preventing and Treating ColonCancer

In order to investigate the effects of the compounds according to anembodiment of the present invention prepared as above to prevent (FIGS.11 and 12 ) and treat (FIGS. 13 and 14 ) colitis-associated colorectalcancer (CA-CRC), medical efficacy was evaluated by administeringCompound 6 to a colorectal cancer model (AOM/DSS mouse).

AOM (Sigma Aldrich, Cat No. A5486) was diluted with physiological salineso as to be a concentration of 10 mg/kg, and then administeredintraperitoneally to C57BL/6 mice (8 weeks old, female, 18±2 g) by 200μl for a total of 3 times at an interval of 7 days (Experiment day 0, 7,14th). On the 7th day of the experiment, a 1.5% DSS solution prepared bydissolving 1.5% DSS in sterile distilled water was given for drinking tothe mice for 7 days. Further, the 1.5% DSS solution was changed at aninterval of 2 days. Sterile distilled water was provided for drinkingfrom the 8th day of the experiment.

In the compound 6 administration group according to an embodiment of thepresent invention, 10 mg/kg of Compound 6 per mouse was completelydissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v/v)of the administered dose, and then, was diluted in phosphate bufferedsaline to prepare the final ethanol:Cremophor EL:phosphate bufferedsaline (0.375:0.375:9.25, v/v/v), followed by oral administration with200 μl of the prepared solution. In order to confirm the preventiveeffect, Compound 6 was administered for a total of 14 times from the 7thto the 20th days of the experiment.

As a result, the control (AOM/DSS+Vehicle) showed a decrease in the bodyweight after 70 days, but the group administered with Compound 6(AOM/DSS+Compound 6) showed an increase in the body weight similar tothat of normal mice (Normal) (FIG. 11 ). In addition, it was confirmedthat the number of tumors was significantly smaller in the compound 6administration group and the size was also significantly smaller thanthe control. That is, it was confirmed that Compound 6 has effects ofpreventing colorectal cancer caused by inflammation (FIGS. 11 and 12 ).

The effects of Compound 6 according to an embodiment of the presentinvention on treating colon cancer was confirmed. From the 50th to the63rd days of the experiment, DSS was administered on the 7th and 14thdays after AOM administration to the mice to induce colon cancer, andCompound 6 was administered 14 times from the 50th to the 63rd days. Asa result, the control (AOM/DSS+Vehicle) showed a decrease in the bodyweight after 70 days, but the group administered with Compound 6(AOM/DSS+Compound 6) showed an increase in the body weight similar tothat of normal mice (Normal) (FIG. 13 ). In addition, it was confirmedthat the number of tumors was significantly smaller in the compound 6administration group and the size was also significantly smaller thanthe control (FIG. 14 ). That is, it was confirmed that Compound 6 waseffective in treating colon cancer (FIGS. 13 and 14 ). Therefore, it wasconfirmed that Compound 6 can be used as an effective preventive andtherapeutic agent for colon cancer.

Example 6: Confirmation of Psoriasis Therapeutic Effect

In order to investigate the therapeutic effects of the compoundsaccording to an embodiment of the present invention prepared as above onpsoriasis, the following experiments were implemented (FIGS. 15 to 17 ).

Hair on the back of female BALB/C mice (8-10 weeks old) was shaved usinga shaver and shaving cream, and 62.5 mg of 5% imiquimod cream wasapplied daily for 6 days to the shaved skin from the next day. Vaselinecream was applied to control mice (Vehicle). In the compound 6administration group according to an embodiment of the presentinvention, 10 mg/kg of the compound per mouse was completely added to anethanol-Cremophor EL mixture (1:1, v/v) corresponding to 15% (v/v) ofthe administered dose, and then, was diluted in phosphate bufferedsaline to prepare the final ethanol:Cremophor EL:phosphate bufferedsaline (7.5:7.5:85, v/v/v), followed by oral administration with 200 μlof the prepared solution daily for a total of 6 times from the 0th tothe 5th days of the experiment. On the 6th day of the experiment, backskin tissues of mice were obtained to perform histology and mRNAexperiments.

As a result, it was confirmed that the epithelial thickness wassignificantly reduced in the compound 6 orally administration group(FIG. 15 ), IL-17a and S100a8 mRNA expression levels which promoteinflammation in skin lesions were significantly reduced (FIG. 16 ), aswell as Foxp3 and IL-10 which induces inhibition of inflammation in skinlesions were markedly increased (FIG. 17 ). Therefore, it was confirmedthat Compound 6 can be used as an effective therapeutic agent forpsoriasis.

Example 7: Confirmation of Graft-Versus-Host Disease Therapeutic Effects

In order to investigate the therapeutic effects of the compoundsaccording to an embodiment of the present invention prepared as above ongraft-versus-host disease, Compound 6 was administered to agraft-versus-host disease model to evaluate its efficacy (FIGS. 18 and19 ).

The spleen of C57BL/6 mice (8 to 12 weeks old, female, 18±3 g) wasexcised, pulverized by adding RPMI medium, and then passed through a 40μm cell strainer (BD Falcon), thereby obtaining a single cellsuspension. The single cell suspension was centrifuged (1200 rpm, 5minutes), and after discarding the supernatant, 1 ml of ACK (ammoniumchloride/potassium bicarbonate) lysis buffer (0.15 M NH4Cl, 1 mM KHCO3,0.1 mM Na2EDTA) was added, followed by stirring for 1 minute and thenwashing the same with RPMI medium. After centrifugation, the cellsuspension was reacted on mouse CD90.2 microbeads (Miltenyi Biotec, CatNo. 130-121-278) at 4° C. for 20 minutes. After complementation of thereaction, the cell suspension was centrifuged, washed with 10 ml ofautoMACS® Running Buffer (Miltenyi Biotec, Cat No. 130-091-221), andthen resuspended with 3 ml of autoMACS® Running Buffer. Then, CD90.2⁺ Tcells were obtained from the cell suspension using Auto MACS pro(Miltenyi Biotec) (positive selection). To obtain bone marrow cells tobe transplanted together with the obtained CD90.2⁺ T cells, both femursand tibias of normal (wild-type) C57BL/6 mice (8-12 weeks old, female,18±3 g) were aseptically acquired. End portions of the femur and tibiawere cut, and the bone marrow was extracted by perfusion of RPMI mediumto the bone tissue with a syringe (femur 21G, tibia 26G). The extractedbone marrow was passed through a 40 μm cell strainer to obtain a singlecell suspension.

The bone marrow single cell suspension was centrifuged, and afterdiscarding the supernatant, 500 μl of ACK lysis buffer was added,followed by stirring for 30 seconds and washing the solution with RPMImedium. After centrifugation, the suspension was reacted on the mouseCD90.2 microbeads at 4° C. for 20 minutes. After complementation of thereaction, the cell suspension was centrifuged, washed with 10 ml ofautoMACS® Running Buffer, and then resuspended with 3 ml of autoMACS®Running Buffer. Then, CD90.2⁻ T cell-depleted bone marrow cells(TCD-BMs) were obtained from the cell suspension through Auto MACS pro(negative selection). The obtained normal CD90.2⁺ T cells and normalTCD-BMs were washed with phosphate buffered saline. T cells wereprepared by resuspending the same in phosphate buffered saline at4×10⁶/ml, while TCD-BM was prepared by resuspending the same inphosphate buffered saline at 5×10⁶/ml.

The normal BDF1 mice (9 to 11 weeks old, female, 19±3 g) were irradiatedwith 950 cGy of radiation divided at an interval of 3 hours by using aradiation irradiator. A graft prepared by mixing the prepared CD90.2⁺ Tcells and TCD-BM at a ratio of 1:1 was injected through the tail vein ofBDF1 mice at a rate of 100 μl. In the compound 6 administration groupaccording to an embodiment of the present invention, 10 mg/kg of thecompound per mouse was completely dissolved in an ethanol-Cremophor ELmixture corresponding to 7.5% (v/v) of the administered dose, and then,was diluted in phosphate buffered saline to prepare the finalethanol:Cremophor EL:phosphate buffered saline (0.375:0.375:9.25,v/v/v), followed by oral administration with 200 μl of the preparedsolution daily for a total of 12 times from the 3rd to 14th days of theexperiment.

The graft-versus-host disease severity index was evaluated at aninterval of 3 to 4 days by visual observation in a severity index systemthat was classified into a total of 10 points with 0 to 2 points foreach item of weight reduction, hair condition, posture, activity andskin change.

As a result of the analysis, the severity index (8±1) ofgraft-versus-host disease occurred in the solvent control (Vehicle) wassignificantly decreased by administration (2.2±1.2) of Compound 6(compared to the solvent control ***, p<0.001, see FIG. 18 ). Throughhistopathological analysis of the colon tissue of mice in eachexperimental group, it was confirmed that the colon tissue inflammatorypathology index was significantly decreased by administration ofCompound 6 (see FIG. 19 ). These results show that Compound 6 can beused as a preventive and therapeutic drug for graft-versus-host disease.

Example 8: Confirmation of Multiple Sclerosis Therapeutic Effect

In order to investigate the therapeutic effects of the compoundsaccording to an embodiment of the present invention prepared as above onmultiple sclerosis, experimental autoimmune encephalomyelitis (EAE) wasinduced in C57BL/6 mice as follows, and Compound 6 was administered tothe mice to evaluate its efficacy (FIG. 20 ).

On day 0 of the experiment, myelin oligodendrocyte glycoprotein (MOG)35-55 peptide (MOG₃₅₋₅₅, Peptron) (200 μg), heat killed Mycobacteriumtuberculosis, Difco, Cat No. 231141) (500 μg), and an adjuvant(incomplete freund's adjuvant, Sigma Aldrich, Cat No. F5506) were mixedfor 7 minutes to form an emulsion. MOG₃₅₋₅₅ emulsion was subcutaneouslyinjected into both flanks of C57BL/6 mice (7-8 weeks old, female),respectively, and then 100 μl of pertussis toxin (Sigma Aldrich, Cat No.P2980) (200 ng) was administered into the tail vein. On the 2nd day ofthe experiment, an equal amount of pertussis toxin was administeredintravenously. Leakage of the emulsion from the injected site of themice was checked by visual observation, so as to confirm the onset ofmultiple sclerosis from the 7th day of the experiment.

In the compound 6 administration group according to an embodiment of thepresent invention, 10 mg/kg of Compound 6 per mouse was completelydissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v/v)of the administered dose, and then, was diluted in phosphate bufferedsaline to prepare the final ethanol:Cremophor EL:phosphate bufferedsaline (0.375:0.375:9.25, v/v/v), followed by oral administration with200 μl of the prepared solution daily for a total of 6 times from the15th to 20th days of the experiment. The multiple sclerosis index wasvisually observed and recorded in the severity index system classifiedinto 0-5 stages.

Autoimmune encephalomyelitis symptoms were indexed according to thefollowing items.

-   -   0: No symptom    -   1: Tail enervated    -   2: Tail enervated, and hindlimb weaken    -   3: Hindlimb paralysis    -   4: Hindlimb paralysis, and forelimb weaken    -   5: Death or being near death

As a result of the analysis, on the 21st day of the experiment, theacute response period, the disease severity index was 2.9±1.92 in thesolvent control (Vehicle) and 0.92±0.58 in the compound 6 administrationgroup, such that the compound 6 administration group showed lowerseverity index (*, p<0.05, FIG. 20 ) than the solvent control (Vehicle),and thereby the therapeutic effects of Compound 6 on multiple sclerosiswas confirmed.

Example 9: Confirmation of Neutrophilic Asthma Therapeutic Effects

In order to investigate the therapeutic effects of the compoundsaccording to an embodiment of the present invention prepared as above onneutrophilic asthma, Compound 6 was administered to a neutrophilicasthma mouse model to evaluate its efficacy (FIG. 21 ).

Sensitization was induced in C57BL/6 mice (7 weeks old, female) byintranasally administering 10 μg of LPS (lipopolysaccharide, SigmaAldrich, Cat No. L2630) and 75 μg of OVA (Ovalbumin, Sigma Aldrich, CatNo. A5503) diluted in phosphate buffered saline (20 μl in total) on the0th, 1st, 2nd and 7th days of the experiment. From the 14th day of theexperiment, 50 μg of OVA diluted in phosphate buffered saline waschallenged by intranasal administration for a total of 8 times at aninterval of 2 days a week until the 36th day. The intranasaladministration was performed after anesthesia using an isofluraneinhalation anesthetic. In the compound 6 administration group accordingto an embodiment of the present invention, 10 mg/kg of Compound 6 permouse was completely dissolved in an ethanol-Cremophor EL mixturecorresponding to 7.5% (v/v) of the administered dose, and then, wasdiluted in phosphate buffered saline to prepare the finalethanol:Cremophor EL:phosphate buffered saline (0.375:0.375:9.25,v/v/v), followed by oral administration with 200 μl of the preparedsolution daily for a total of 10 times from the 27th to the 36th days ofthe experiment.

As a result, the number of neutrophils in the bronchoalveolar lavage(BAL) was significantly decreased in the compound 6 administration groupcompared to solvent control (Vehicle) (FIG. 21 ), and thereby thetherapeutic effects of Compound 6 on neutrophilic asthma was confirmed.

Example 10: Comparison of Effects Between the Inventive Compounds andComparative Example 1

In order to confirm the superiority of the compounds of the examplesaccording to an embodiment of the present invention compared with thecompound of Comparative Example 1 (briefly, “Comparative Example 1”),the following experiments were implemented: (1) measurement ofpharmacokinetic parameters; (2) measurement of solubility; (3)confirmation of inhibitory effects on Th17 cell inflammatory activity;(4) confirmation of promotion effects of the production ofFoxp3-expressing regulatory T cells; (5) confirmation whether to inhibitactivity of drug metabolism-related enzyme; and (6) confirmation of thetherapeutic effects of inflammatory bowel disease.

(1) Measurement of Pharmacokinetic Parameters

Pharmacokinetic parameters of Compound 6 and Comparative Example 1 weremeasured (Table 3, and FIG. 22 ).

For rats, an oral administration group was fasted for 16 hours, and anintravenous administration group was subjected to intravenousadministration with 10 mL/kg of solution prepared by completelydissolving Compound 6 in an ethanol-Cremophor EL mixture correspondingto 7.5% (v/v) of the administered dose, and then diluting in phosphatebuffered saline so as to be the final ethanol:Cremophor EL:phosphatebuffered saline (0.375:0.375:9.25, v/v/v), without fasting, at the doseconcentration listed in Table 3. The oral administration group wassupplied with food about 4 hours after administration. Blood collectionwas performed on the intravenous administration group after 0.083, 0.5,1, 2, 4, 8, 24 hours from the administration and on the oraladministration group after 0.5, 1, 2, 4, 8, 12, 24 hours from theadministration, and plasma was recovered using centrifugation. In therecovered plasma, the drug concentration in the plasma was measuredusing UHPLC-MS/MS method.

As listed in Table 3, it was confirmed that, in the case of orallyadministering Compound 6 at a concentration of 10 mg/kg, thebioavailability (F %), the AUClast and Cmax were increased 1.9, 5.9 and11.9 times, respectively, compared to the case of orally administeringComparative Example 1 at a concentration of 10 mg/kg. In addition, itwas confirmed that, in the case of orally administering Compound 6 at aconcentration of 50 mg/kg, the bioavailability (F %), the AUClast andCmax were increased 67.2, 66.8 and 30.2 times, respectively, compared tothe case of orally administering Comparative Example 1 at aconcentration of 100 mg/kg. From the above results, it was confirmedthat Compound 6 could exhibit drug efficacy even in a small amountcompared to Comparative Example 1.

TABLE 3 Administration compound Comparative Example 1 Compound 6 SectionG1 G2 G3 G4 G5 G6 Administration 10 100 1 10 50 1 concentration (mg/kg)Administration Oral Oral Intra- Oral Oral Intra- route (PO) (PO) venous(PO) (PO) venous (IV) (IV) Cmax or C0 126.2 151.9 405.7 741.67 4590.001422.77 (ng/mL) Tmax (hr) 1.0 0.5 — 2.00 4.00 — t1/2 (hr) 0.9 6.8 0.82.0 3.83 1.04 AUClast 366.0 644.2 183.1 4346.02 43059.37 1164.82 (ng ·hr/mL) CL (mL/hr/kg) — — 430.5 — — 862.26 Vdss (mL/kg) — — 310.7 — —923.76 Dose ratio 1.0 10.0 — 1 5 — Cmax ratio 1.0 1.2 — 1.0 6.2 —AUClast Ratio 1.0 1.8 — 1.0 9.9 — Bioavailability 20.0 1.1 — 37.3 73.9 —(F %)

(2) Measurement of Solubility

The solubility of Compound 6 and Comparative Example 1 was measured.

After dissolving Compound 6 in ethanol, the solubility was measured andanalyzed using a SIRIUS T3 machine.

As listed in Table 4, it was confirmed that the logP value of Compound 6was greater than that of Comparative Example 1, and the pH-metricsolubility molarity was about 3.92 times.

TABLE 4 pH-metric results Section Comparative Example 1 Compound 6 logP2.80 3.04 Molarity 3.65 μM 14.3 μM Weight/ml  1.248 μg/ml  6.104 μg/ml

(3) Confirmation of Inhibitory Effects on Th17 Cell InflammatoryActivity

The inhibitory effects of Compound 6 and Comparative Example 1 on theinflammatory activity of Th17 cells, which are key causing cells ofautoimmune disease, were compared (FIG. 23 ).

The spleen of C57BL/6 mice (8 to 12 weeks old, female) was excised,pulverized by adding IMDM medium, and then passed through a 40 μm cellstrainer (BD Falcon), thereby obtaining a single cell suspension. Thesingle cell suspension was centrifuged (1200 rpm, 5 minutes), and afterdiscarding the supernatant, 1 ml of ACK lysis buffer was added, followedby stirring for 1 minute and then washing the same with RPMI medium.After the single cell suspension was centrifuged, T-cells were separatedusing a Mouse CD4 Naive T cell Enrichment Kit (Invitrogen). Theseparated T-cells were prepared by resuspending them in RPMI-fetalbovine serum (FBS) 10%+2-ME (mercaptoethanol) medium at 1×10⁶/ml. ForT-cell activation, 5 μg/ml anti-CD3 (eBioscience™) was dispensed by 100μl onto a 96-well plate, reacted in a cell incubator (37° C., 5% CO₂incubator) for 4 hours, and washed with phosphate buffered saline toprepare the plate. The T-cells were dispensed by 200 μl onto theprepared plate, and each well was treated with 2 μg/ml of anti-CD28(eBioscience™), 3 ng/ml of TGFβ-1 (R&D systems), 10 ng/ml of IL-2 (R&Dsystems), 5 μg/ml of anti-mouse IFN-γ (Bio X cell), and 5 μg/ml ofanti-mouse IL-4 (Bio X cell). Each 5 μl of compounds at concentrationsof 1.0 μM, 2.5 μM and 5 μM diluted in RPMI-fetal bovine serum 10%+2-MEmedium was used for treatment, followed by culturing the same in a cellincubator (37° C., 5% CO₂ incubator) for 3 days. As a control, 5 μl of0.05% dimethylsulfoxide (DMSO)/RPMI medium was used for treatment. After3 days, in order to confirm inhibitory effects of the compounds on theinflammatory activity of Th17 cells, the cultured medium was recoveredto confirm IL-17A with flex set (BD biosciences)

To confirm IL-17A cytokine, 25 μl of the recovered medium and 25 μl ofassay diluent buffer were put in FACS tubes (BD falcon), and the samplewas diluted 1 to 2 (½). 1 μl of capture bead was put into 49 μl ofcapture bead diluent to prepare 50 μl of capture bead solution persample. After mixing the capture bead solution by vortexing, it wasadded to the FACS tubes containing each sample by 50 μl, followed byvortexing again, and left at room temperature for 1 hour. After 1 hour,1 μl of PE detection reagent was added to 49 μl of PE detection reagentdiluent to prepare 50 μl of PE detection solution per sample. Aftervortexing the PE detection solution, it was added to the FACS tubescontaining the capture bead solution and the sample by 50 dl. Aftervortexing the FACS tubes, they were placed at room temperature for 1hour. After 1 hour, 1 ml of CBA wash buffer was added per tube, andcentrifugation was performed at 400 g for 5 minutes, then thesupernatant was removed. After weakly vortexing, 150 μl of Fix bufferwas added and weakly vortexed, followed by analysis using flowcytometry.

As a result, it was confirmed that IL-17A production of Th17 cells wassignificantly decreased by Compound 6 compared to Comparative Example 1(FIG. 23 ).

(4) Confirmation of Promotion Effects of the Production ofFoxp3-Expressing Regulatory T Cells (Treg)

The promotion effects of Compound 6 and Comparative Example 1 on theproduction of “FoxP3-expressing regulatory T cells,” which play animportant role in maintaining immune tolerance, were compared (FIG. 24). CD4⁺ T cells were separated and cultured in the same experimentalmethod as in FIG. 4 , then treated with each 5 μl of compounds atconcentrations of 2.5 NM and 5 μM diluted in RPMI-fetal bovine serum10%+2ME medium. After culturing in a cell incubator (37° C. 5% CO₂incubator) for 3 days, analysis was performed. As a control, 5 μl of0.05% dimethylsulfoxide (DMSO)/RPMI medium was treated. After 3 days, inorder to confirm the effects of production of the regulatory T cells,the cultured cells were recovered and Foxp3 protein expression wasmeasured in the same manner as in FIG. 4 .

As a result, it was confirmed that the production of Foxp3-expressingregulatory T cells was significantly increased by Compound 6 compared toComparative Example 1 (FIG. 24 ).

(5) Confirmation Whether to Inhibit Activity of Drug Metabolism-RelatedEnzyme

The activity of CYP isoenzyme, which is a drug metabolism-relatedenzyme, was measured.

Live human hepatocyte cell lines (Corning, Cat No. 454551) werecollected using a plating medium, seeded on a 24-well plate so that acell density thereof was 0.4×10⁶ cells/well, and cultured for 2 to 4hours. After replacing the plating medium with a hepatocyte culturemedium, the hepatocytes were cultured for 24 hours. After culturing for24 hours, hepatocytes were treated with Compound 6 at 1, 5, 25 M. Themedium containing the compound was changed every 24 hours for 2 days.The cultured cells were recovered to prepare mRNA samples. mRNA wasextracted from the recovered cells by a phenol-chloroform sedimentationmethod using TRIZOL reagent (Invitrogen, Cat No. 15596018). From theisolated RNA, cDNA was synthesized by reverse transcription, followed bymeasuring drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2B6, andCYP3A4 through real-time PCR analysis using the Quant Studio™ 7 FlexReal-Time PCR System (Applied Biosystems, CA). Relative values of theenzyme expression levels were compared by the ΔΔct method using GAPDH asa control enzyme. Herein, one (1) fold was set using the control.Results are expressed as % of control compared to control sample.

Table 5 illustrates changes in CYP isoenzyme activity after 10 μMtreatment of each compound (Comparative Example 1, Compound 6,Ketoconazole) (% of Control Activity)

TABLE 5 Section CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Comparative 64.691.9 24.4 93.3 107.6 Example 1 Compound 6 88.2 94.5 93.1 101.2 85.7Ketoconazole 95.4 98.6 96.7 97.9 34.7 (0.1 μM, CYP3A4 inhibitor)

As a result, it was confirmed that Comparative Example 1 inhibited theenzyme activity of CYP2C19 by 24.4% at 10 μM, whereas Compound 6 hadlittle effect on the enzyme activity of CYP2C19. That is, it wasconfirmed that Compound 6 could be administered in combination withdrugs metabolizing CYP2C19.

(6) Confirmation of the Therapeutic Effects of Inflammatory BowelDisease

The therapeutic effects of Compound 6 and Comparative Example 1 oninflammatory bowel disease were compared. Inflammatory bowel disease wasinduced in C57BL/6 mice in the same manner as in Example 4, and afteroral administration of 10 mg/kg of Compound 6 and Comparative Example 1to mice, the expression levels of IL-6 and S100a9 mRNA were confirmed incolon tissues.

As a result, it was confirmed that the expression levels of IL-6 andS100a9 mRNA in the colon tissues were significantly decreased byCompound 6 compared to Comparative Example 1 (FIG. 25 ). That is, it wasconfirmed that Compound 6 showed a higher therapeutic effect ofinflammatory bowel disease than Comparative Example 1.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

A sequence listing electronically submitted on Apr. 25, 2023, as a XMLfile named 20230425_LC0532305_TU_SEQ.XML, created on Apr. 20, 2023 andhaving a size of 25,861 bytes, is incorporated herein by reference inits entirety.

What is claimed is:
 1. A compound represented by Formula 1 below, astereoisomer thereof or a pharmaceutically acceptable salt thereof:

wherein, A is hydrogen, halogen, a hydroxyl group, a C₁-C₃ alkyl group,a C₂-C₃ alkenyl group, a C₂-C₃ alkynyl group, a C₁-C₃ alkoxy group,dimethylamine, —NO₂, —CN, —COOR₂ or —S(═O)₂R₂, B is hydrogen, a C₁-C₃alkyl group, a phenyl group, an acetyl group, —CH₂C(═O)OR₂, —C(═O)OR₂ or—S(═O)₂R₂, R₁ is a substituted or unsubstituted 5-7 memberedheterocyclic ring or —NH₂, and A R₂ is a C₁-C₃ alkyl group.
 2. Thecompound, the stereoisomer or the pharmaceutically acceptable saltthereof according to claim 1, wherein the substituted 5-7 memberedheterocyclic ring is a 5-7 membered heterocyclic ring substituted with aC₁-C₃ alkyl group, a hydroxyl group or dimethylamine.
 3. The compound,the stereoisomer or the pharmaceutically acceptable salt thereofaccording to claim 1, wherein the substituted or unsubstituted 5-7membered heterocyclic ring is any one selected from the group consistingof the following heterocyclic rings:


4. The compound, the stereoisomer or the pharmaceutically acceptablesalt thereof according to claim 1, wherein the compound represented byFormula 1 is any one selected from the group consisting of the followingcompounds:


5. A pharmaceutical composition comprising the compound, thestereoisomer or the pharmaceutically acceptable salt thereof accordingto claim
 1. 6. A method for treatment of autoimmune disease, the methodcomprising administering the pharmaceutical composition according toclaim 5 to a subject in need thereof.
 7. The method according to claim6, wherein the autoimmune disease is any one selected from the groupconsisting of inflammatory bowel disease, multiple sclerosis,graft-versus-host disease, asthma, and psoriasis.
 8. A method fortreatment of colon cancer, the method comprising administeringpharmaceutical composition according to claim 5 to a subject in needthereof.